mined by its overall polarity, the polarity of the stationary phase and the nature
of the sample components. Unlike a GCcarrier gas, which plays no part in
chromatographic retention and selectivity, the composition of an HPLCmobile
phase is crucial in both respects. For normal-phaseseparations (stationary
phase morepolar than mobile phase), eluting power increaseswith increasing
solvent polarity, whilst for reversed-phaseseparations (stationary phase less
polar than mobile phase), eluting power decreaseswith increasing solvent
polarity. An eluotropic series of solvents, which lists them in order of
increasing polarity, is a useful guide to solvent selection for HPLC separations.
Table 1 is an example that also includes UV cut-off wavelengths as UV
absorbancedetectors are the most widely used (vide infra). Elution can be under
isocratic conditions (constant mobile phase composition) or a composition
gradient can be generated by a gradient formerto improve the resolution of
complex mixtures, especially if the sample components have a wide range of
polarities. The most widely used mobile phases for reversed-phase separations
are mixtures of aqueous buffers with methanol, or water with acetonitrile. For
normal-phase separations, which are less common, hydrocarbons blended with
chlorinated solvents or alcohols are typical.Table 1. An eluotropic series of solvents for HPLC
Solvent Solvent strength Solvent strength UV cut-off
parameter, e° parameter, p¢ (nm)
(adsorption) (partition)
n-Hexane 0.01 0.1 195
Cyclohexane 0.04 -0.2 200
Tetrachloromethane 0.18 1.6 265
Methylbenzene 0.29 2.4 285
Trichloromethane 0.40 4.1 245
Dichloromethane 0.42 3.1 230
Tetrahydrofuran 0.56 4.0 212
Propanone 0.56 3.9 330
Acetonitrile 0.65 5.8 190
iso-Propanol 0.82 3.9 205
Ethanol 0.88 4.3 205
Methanol 0.95 5.1 205
Ethanoic acid > 1 4.4 255
Water > 1 10.2 170The mobile phase is either a single solvent or a blend of two to four solvents
delivered at pressures of up to about 5000 psi (350 bar) with a constant and
reproducible flow rate of <0.01–5 cm^3 min-^1. The solvent delivery system
comprises the following components:● A mechanical pumpdesigned to deliver a pulse-freeflow of mobile phase.
Most are single or dual piston reciprocating pumps(Fig. 2) with specially
designed cams and pulse dampers to minimize or eliminate inherent flow
variations, or one-shot pulseless syringe pumpsused primarily with micro-
bore columns (vide infra) requiring low flow rates. The wetted parts of the
pump should be inert to all solvents (stainless steel, titanium, industrial
sapphire, ruby, and Teflon being the principal choices) with minimal volume
pumping chambers to facilitate rapid changes of mobile phase composition.Solvent delivery
system
D6 – HPLC: principles and instrumentation 157